We developed a novel method for recording extracellular neural signal from the brain of freely swimming fish. This opens the door to studying the neuron mechanisms at end of day fish behavior. This method is valid and water proof and therefore allows recording while the fish swim freely in the water tank.
In addition, the recording location can be controlled by the built in micro drive. Tal Zoor-Novoplansky, our lab technician, will help me demonstrate this procedure. To construct the housing, use a brass plate with dimensions of 19 millimeters by 29 millimeters by one millimeter.
With two 5.5 millimeter slits on the long sides, perpendicular to the edge. The slits should be 6.5 millimeters away from the narrow side. Using pliers, fold the area between the slits on the long sides inward.
Then fold the bottom part inward and the upper side outward to obtain the housing. Next, use a three millimeter dill bit to make holes in the micro drive housing for the screws and solder the sides of the housing. Use a fine circular file to create a small semi circular slit at the bottom of the housing that has a radius of 1.5 millimeters.
Using a one millimeter drill bit make a hole in the back of the housing for the tetrodes. First, use a cutter to break off a three pin piece from a single row male pin header strip. Using pliers pull out the middle pin.
Next, use a cutter to cut the remaining pins to 10 millimeters in length. Use a number 65 drill bit to drill a hole through the middle pin hole and use a double zero 99 tap to drill a thread. Assemble the micro drive and the brass plates.
Insert a screw through the first brass plate, through the pin header thread and then through the second brass plate. Finally, place a nut on the screw and gently tighten the assembled micro drive. Solder the pins together with the brass plates and solder the nut together with the tip of the screw.
After this, solder the micro drive into the micro drive housing at four points on the sides of the micro drive brass plates. Using epoxy glue a six millimeter long stainless steel tube to the small semi circular slit at the bottom of the micro drive housing. Glue a three millimeter long stainless steel tube to the pin head so that it is lined up with the six millimeter long tube on the housing.
Insert the prepared silicone and polyimide tubes into the two stainless steel tubes and use cyanoacrylate glue to glue them to the stainless steal tube attached to the pin header. Then, screw the micro drive all the way up and cut off the excess tubing from the top and bottom of the two steel tubes. Next, cut two bare silver wires with a diameter of 75 micrometers to a length of 12 centimeters and solder them both to the ground connection in the electrode interface board and to all unused channels.
Push one of the prepared tungsten wires into one of the holes of a 16 channel electrode interface board. Place a pin into the hole and press it with a pair of pliers. Measure the resistance between the pin and the un-coded side of the wire to check for connectivity.
Repeat this process for all of the prepared wires including one 50 micrometer wire for reference electrode. Then group the electrode wires into two groups of four wires each and tape them together using duct tape at the end of each wire leaving the reference wire alone. Hold the electrode interface board above a motorized turning device and place the duct tape end from one group of four wires on the device.
Apply 130 rounds clockwise followed by 20 counterclockwise rotations. Then, apply cyanoacrylate glue to cover the tetrode. After the glue has cured, cut the tetrode close to the duct tape.
Repeat this rotating, gluing and cutting process to the other tetrode. First, attach the micro drive housing to the logger box with two millimeter screws. Thread the tetrodes and the reference electrode through the hole at the back of the micro drive housing.
Thread the tetrodes through the two silicone tubes and thread the 50 micrometer tungsten wire through the polyimide tube. Screw the micro dive all the way down and apply cyanoacrylate glue to the top of the tubes to glue the tetrodes and wires to their tubes and ensure movement is consistent with the micro drive. Screw the micro drive all the way to the top.
Apply soft petroleum to the exposed tetrode and wires inside the micro drive housing to prevent motion. Attach a precut petri dish bottom window to the front of the micro drive housing with epoxy while making sure to keep the ground wires outside the window. Next, apply room temperature vulcanizing coating to the exposed tetrodes and wires between the logger box cover and the microdrive housing.
Use sharp scissors to cut the tetrodes and reference wire to the desired length. After this attach the marked, exuded polystyrene foam to the box. Adjust its size so its buoyancy is balanced when submerged in the water bath.
After anesthetizing the fish, take the fish out of the water and place it in the holder. Use a sterile spatula to apply five percent lidocaine paste on the skin above the designated surgery site for 10 minutes. Then remove the lidocaine.
Use a sterile number 15 blade scalpel to remove the skin above the skull in the region designated for the implant. Then use a dental drill with 0.7 millimeter drill bits to drill four holes in the skull. Apply cyanoacrylate glue to one of the holes and immediately insert a one millimeter screw.
Repeat this process for the remaining drill holes. After this, use a dental burnisher to apply dental cement on the screws and on the periphery of the exposed skull. Using the dental drill make a hole that is five millimeters in diameter above the brain region of interest.
Use fine tweezers and soft tissue paper to remove fatty tissue between the skull and brain and expose the brain region target being careful to not damage the large blood vessels underneath the skull. First, lower the implant such that the electrodes are inserted into the brain while the bottom part of the micro drive housing is near the skull. Start attaching the implant to the skull by applying a small quantity of dental cement between the housing and the nearest skull screw.
After the first part of the dental cement is cured apply addition dental cement and close the hole above the skull and the entire exposed skull. After flushing the fish's gills with fresh water to wake it, remove the fish from the holder and place it back in its home tank. Make sure the fish is able to swim freely with the implant.
The goal of these experiments is to study how the neural activity of single cells encodes the fish's behavior. To accomplish this a tetrode array is implanted into the telencephalon of freely swimming goldfish which allows for the neural activity to be recorded. The brain activity while being recorded is digitized at 31, 250 Hertz and high pass filtered at 300 Hertz by the data logger.
Then offline a ban pass filter is applied to the signals and the presorted raw data are separated into each tetrode's channel and the reference channel. Next common spike sorting algorithms are used to characterize single cell activity. First each channel is manually filtered by the minimal spike amplitude threshold.
Spikes that appeared in more than one tetrode or in the reference channel are also filtered. The detected spikes are then manually clustered and filtered by shape, length, inter spike interval and principle component analysis. It is important to check the implant integrity before the surgery.
The tetrode can be checked using a standard impedance meter and the box should be checked for leakage underwater. This method can be modified to work with many aquatic animals as long as they big enough to hold the system. This enables to study the neural mechanism that underlie their behavior.
